We have evolved several independent populations of methicillin-sensitive as well as methicillin-resistant S. aureus (MRSA) to multiple fluoroquinolones, and identified the adaptive trajectories leading to antibiotic resistance. Apart from mutations in previously implicated as well as uncharacterized regions of target proteins, we also found mutations in novel genes involved in metabolism, transcriptional regulation, and efflux. Several of these mutations arose early during the evolution, before those in target proteins. We are characterizing how these mutations lead to resistance, and whether altering persistence levels impacts early steps of resistance evolution. We are also analyzing adaptive paths leading to increased persistence in multiple evolved populations. In the future we will test the importance of the identified mutations in clinical antibiotic resistance. We will also utilize transposon libraries to identify additional genes that play a role in antibiotic resistance and persistence.